Purification and characterization of liver cytochrome P-446 isolated from protein energy malnourished rats

A liver cytochrome P-450 isozyme has been purified to homogeneity from protein-energy malnourished rats induced with -naphthoflavone (-NF). The purification steps included chromatography on DEAE-Sephadex-A-25, DEAE-cellulose (DE-53), hydroxylapatite (HA) and carboxymethyl-sephadex (CM) columns. The reduced carbon monoxide difference and absolute spectra showed a Soret peak at 446.5 nm. The wavelength maxima for the oxidized and reduced spectra were at 416 and 408 nm, respectively. Cytochrome P-446 appears to have a predominantly low spin ferric iron, migrates as a single band of molecular weight 56000 in sodium dodecyl sulfate polyacrylamide gels and has a specific content of 14 nmol/mg of protein. P-446 oxidized various substrates at different rates in a reconstituted system with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine. In this system turnover rates for benzo[]pyrene, testosterone and benzphetamine oxidation were: 81.10; 1.85 and 1.42 nmoles product/min/nmol P-446 respectively. While NH₂ terminal amino acid sequence analysis of 18 of the first 20 residues suggests that the cytochrome P-446 isolated from malnourished rats is identical with form c, the catalytic activities suggest that this isozyme may be a more effective or efficient catalyst for some substrates.Abbreviations -NF -napthoflavone - SDS-PAGE Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis - 3-MC 3-Methyl Cholanthrene - PEG Poly Ethylene Glycol - DTT Dithiothreitol - PMSF Phenyl Methyl Sulfonylfluoride - EDTA disodium ethylenediaminetetraacetate - NADPH reduced nicotinamide adenine dinucleotide phosphate - P-450 cytochrome P450, PB-1, PB-4, PB-5 and P-450 isozymes purified from phenobarbital induced rat liver - HPLC High Pressure Liquid Chromatography - B[]P benzo[]pyrene - CM Carboxymethyl Sephadex - PTH-amino acid phenylthiohydantoin amino acid, Cytochrome P-450 EC 1.14.14.1, NADPH Cytochrome P-450 (c) reductase ED 1.6.2.4     

Purification and characterization of cytochrome P-450 isozymes from phenobarbital-induced adult hen liver

1. Two cytochrome P-450 isozymes (P-450 PB-A, PB-B) and cytochrome b5 were purified from livers of phenobarbital-treated adult hens. 2. Both the enzymes exhibited the same apparent molecular weight (54,000). 3. They could be distinguished on the basis of immunochemical properties, spectral properties, peptide pattern after partial proteolysis, tryptic peptide pattern, and N-terminal sequence. 4. The antibodies raised against P-450 PB-A and PB-B did not cross-react with microsomal P-450s of rat, mice, cat, or catfish species by immunoblotting.     

Purification of human liver cytochrome P-450 and comparison to the enzyme isolated from rat liver

Human liver cytochrome P-450 was isolated from autopsy samples using cholate extraction and chromatography on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE-cellulose gels. Purified preparations contained as much as 14 nmol cytochrome P-450 mg^?1 protein, were free of other hemoproteins, and were active in the mixed-function oxidation of d-benzphetamine and 7-ethoxycoumarin when coupled with either rat or human liver NADPH-cytochrome P-450 reductase. Some of the preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; apparent subunit M_rs estimated for several preparations were 53,000 or 55,500. The amino acid composition of one preparation was determined and found to resemble those of rat liver cytochromes P-450, although some variations were noted. Rabbit antibodies raised to phenobarbital-treated rat liver cytochrome P-450 were more effective in inhibiting d-benzphetamine N-demethylase activity in human liver microsomes than were antibodies raised to 3-methylcholanthrene-treated rat liver cytochrome P-450. These antibodies also inhibited benzo(a)pyrene hydroxylation in human liver microsomes, although the inhibition patterns did not follow a general pattern as in the case of benzphetamine demethylase activity. Microsomes prepared from three different human liver samples were more effective in eliciting complement fixation with antibodies raised to phenobarbitalthan to 3-methylcholanthrene-treated rat liver cytochrome P-450. Complement fixation in such systems appears to result from similarity of certain rat and human liver cytochrome P-450 antigenic determinants, as fixation could be inhibited by removal of cytochrome P-450-directed antibodies from the total immunoglobulin population and purified human cytochrome P-450 was more effective (on a protein basis) than liver microsomes in producing fixation. Human liver microsomes prepared from five different individuals all produced ≥ 90% complement fixation, but variations were observed in the fixation curves plotted either versus microsomal protein or versus spectrally detectable microsomal cytochrome P-450.These results indicate that human liver microsomal cytochromes P-450 can be isolated using modifications of techniques developed for laboratory animals and that human and rat liver cytochromes P-450 share certain features of structural, functional, and immunological similarity. The available data suggest the existence of multiple forms of human liver microsomal cytochrome P-450, but possible artifacts associated with the use of autopsy samples suggest caution in advancing such a conclusion.     

Purification and characterization of diabetes-inducible cytochrome P-450

A diabetes-inducible form of cytochrome P-450, termed P-450DM, was purified to electrophoretical homogeneity (MW 51,000) by high-performance liquid chromatography from liver microsomes of diabetic rats induced with streptozotocin. The CO-reduced absorption maximum of P-450DM was at 452 nm and the oxidized heme iron appeared to be predominately in the high-spin state as deduced from the Soret maximum at 395 nm. P-450DM was active in aniline hydroxylation and N-nitrosodimethylamine demethylation. The dealkylation activity toward 7-ethoxycoumarin by P-450DM was much enhanced by the addition of cytochrome b5.     

Purification of a new cytochrome P-450 from human liver microsomes

Using a classical methodology of purification consisting of three chromatographic steps (Octyl-Sepharose, DEAE-cellulose, CM-cellulose) we have purified a new cytochrome P-450 from human liver microsomes. It was called cytochrome P-450(9). It has been proven to be different from all precedingly purified human liver microsomal cytochrome P-450 isozymes by its immunological and electrophoretical properties. It does not cross-react with any rat liver cytochrome P-450 and anti-cytochrome P-450(9) does not recognize rat liver microsomes; thus this cytochrome P-450(9) is specific to humans. This cytochrome P-450 isozyme exists in low amounts in human liver microsomes and exhibits an important quantitative polymorphism. In reconstituted system, cytochrome P-450(9) is able to hydroxylate all substrates tested but is not specific of any; its exact role in xenobiotic metabolism in man remains to be elucidated.     

Purification and characterization of cytochrome P-450-dependent arachidonic acid epoxygenase from human liver

A novel human liver cytochrome P-450 isozyme (P-450-AA), which catalyzes arachidonic acid epoxidation, has been purified to electrophoretic homogeneity from human liver. As judged spectrally, the newly described isozyme is low spin in the oxidized state, with a soret band at 415 nm and an increased maximum at 451 nm in the CO-difference spectrum. Cytochrome P-450-AA appeared homogeneous as judged by the appearance of a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an estimated molecular weight of 53,100. Although cytochrome P-450-AA had a relatively low specific content of 10.8 nmol/mg, it possessed a high activity of arachidonic acid epoxidation. The P-450-AA oxidized arachidonic acid in a reconstituted system into the four regioisomeric epoxyeicosatrienoic acids (EETs) (5, 6-, 8, 9-, 11, 12-, 14, 15-EETs) at a rate of 2,010 pmol/nmol/min, a rate which is 37-fold higher than that observed with the crude microsomal preparation. Moreover, the purified cytochrome P-450-AA catalyzed the de-ethylation of 7-ethoxyresorufin at the rate of 2970 pmol/nmol/min, whereas other cytochrome P-450-dependent reactions were carried out at 23-2,000-fold lower rates and ranged between 0.3-130 pmol/nmol/min. The amino acid composition is different from that of other cytochrome P-450 isozymes. The NH2-terminal sequence of 20-amino acid residues was compared to that of LM2 and PB2-B2, the phenobarbital-induced forms in rabbit and rats, respectively. Comparison was also made with two forms of human cytochrome P-450, HLc and HLd. There were 7/20 identical residues for P-450-AA and LM2 and 4/20 for P-450-AA and PB2-B2. There were 2/20 identical residues for P-450-AA and HLd, and no identical residues were found for HLc. We conclude that the biologically active EETs, are formed by a distinct and unique P-450 isozyme from human liver and that arachidonic acid can serve as a screen for detection of the novel P-450 isozyme.     

Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver

We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.     

Evidence that isoniazid and ethanol induce the same microsomal cytochrome P-450 in rat liver, an isozyme homologous to rabbit liver cytochrome P-450 isozyme 3a

Cytochrome P-450j has been purified to electrophoretic homogeneity from hepatic microsomes of adult male rats administered ethanol and compared to the corresponding enzyme from isoniazid-treated rats. The enzymes isolated from ethanol- and isoniazid-treated rats have identical chromatographic properties, minimum molecular weights, spectral properties, peptide maps, NH2-terminal sequences, immunochemical reactivities, and substrate selectivities. Both preparations of cytochrome P-450j have high catalytic activity in aniline hydroxylation, butanol oxidation, and N-nitrosodimethylamine demethylation with turnover numbers of 17-18, 37-46, and 15 nmol product/min/nmol of P-450, respectively. A single immunoprecipitin band exhibiting complete identity was observed when the two preparations were tested by double diffusion analysis with antibody to isoniazid-inducible cytochrome P-450j. Ethanol- and isoniazid-inducible rat liver cytochrome P-450j preparations have also been compared and contrasted with cytochrome P-450 isozyme 3a, the major ethanol-inducible isozyme from rabbit liver. The rat and rabbit liver enzymes have slightly different minimum molecular weights and somewhat different peptide maps but similar spectral, catalytic, and immunological properties, as well as significant homology in their NH2-terminal sequences. Antibody to either the rat or rabbit isozyme cross-reacts with the heterologous enzyme, showing a strong reaction of partial identity. Antibody against isozyme 3a specifically recognizes cytochrome P-450j in immunoblots of induced rat liver microsomes. Aniline hydroxylation catalyzed by the reconstituted system containing cytochrome P-450j is markedly inhibited (greater than 90%) by antibody to the rabbit protein. Furthermore, greater than 85% of butanol or aniline metabolism catalyzed by hepatic microsomes from ethanol- or isoniazid-treated rats is inhibited by antibody against isozyme 3a. Results of antibody inhibition studies suggest that cytochrome P-450j is induced four- to sixfold by ethanol or isoniazid treatment of rats. All of the evidence presented in this study indicates that the identical cytochrome P-450, P-450j, is induced in rat liver by either isoniazid or ethanol, and that this isozyme is closely related to rabbit cytochrome P-450 isozyme 3a.     

Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450tu) from mouse liver tumors

Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15 alpha-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent Mr of 50,000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15 alpha-hydroxylation of progesterone (2.3 min-1) and testosterone (2.5 min-1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450(15) alpha, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450tu differed from P-450(15) alpha by its higher molecular weight, its 40-times lower steroid 15 alpha-hydroxylation and its 4-times higher benzphetamine N-demethylation.     

Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse liver

Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome . CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a V twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase, or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated 'P-450Coh'. Anti-(P-450Coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of about 51 kDa. The immunoprecipitated translation products correspond to a messenger RNA of 2100 +/- 100 nucleotides.     

Purification and characterization of rat pulmonary cytochrome P-450

The pulmonary cytochrome P-450, P450 L-2, was purified 460-fold from pulmonary microsomes of untreated male rats. Its specific content was 10.6 nmol/mg of protein. The monomeric molecular weight was 54,000 on SDS-polyacrylamide gel electrophoresis. The CO-reduced absorption maximum of P450 L-2 was at 451 nm, and the oxidized heme iron appeared to be in the low-spin state, as deduced from the Soret maximum at 421 nm. P450 L-2 had high lauric acid omega- and (omega-1)-hydroxylation activities, but low prostaglandin A1 omega- and (omega-1)-hydroxylation activities. It catalyzed the O-dealkylation of 7-ethoxycoumarin, but was not efficient in the hydroxylation of testosterone or the N-demethylation of aminopyrine. The NH2-terminal amino acid sequence of P450 L-2 was V-L-N-F-L-X-P-X-L (X being an unidentified residue). The catalytic properties of P450 L-2 resembled those of P450 K-5, the major rat renal cytochrome P-450. However, anti-P450 K-5 antibody did not cross-react with P450 L-2, and these forms had different NH2-terminal sequences. To judge from the results of NH2-terminal sequence analysis, P450 L-2 seems to be placed in the IVB gene family. Also, P-450 IIB1 was detected by immunoblotting in one of the peaks on ion-exchange HPLC during the purification of P450 L-2, suggesting the presence of P-450 IIB1 in rat pulmonary microsomes.     

Purification and characterization of seven distinct forms of liver microsomal cytochrome P-450 from untreated and inducer-treated male Wistar rats

A total of nine forms of cytochrome P-450 were purified to homogeneity from liver microsomes of male Wistar rats. They were P-451 I and P-451 II from untreated rats, P-450 II and P-450 III from phenobarbital-treated rats, MC-P-448 L and MC-P-448 H from 3-methylcholanthrene-treated rats, and P-452, P-448 L, and P-448 H from 3,4,5,3',4'-pentachlorobiphenyl-treated rats. Among them, MC-P-448 L and MC-P-448 H were indistinguishable from P-448 L and P-448 H, respectively, with regard to electrophoretic, spectral, catalytic and immunochemical properties, and thus seven forms were distinct hemoproteins. The minimal molecular weight of each form was as follows: P-451 I (49,000), P-451 II (52,000), P-450 II (52,000), P-450 III (53,500), P-452 (48,000), P-448 L (56,000), P-448 H (54,000). Judging from the oxidized absolute spectra, P-448 H was a high-spin form and the others were of low-spin type. In a reconstituted system, N-demethylations of benzphetamine and aminopyrine were catalyzed by most of the forms at comparable rates. On the other hand, the activities for the oxidations of benzo[a]pyrene, 7-ethoxycoumarin, biphenyl, and estradiol-17 beta varied greatly among the forms of cytochrome P-450. The most efficient catalysts were as follows: P-448 L and P-451 II for benzo[a]pyrene 3-hydroxylation; P-448 L for 7-ethoxycoumarin O-deethylation; P-448 L, P-451 II, and P-448 H for biphenyl 4-hydroxylation; P-448 L and P-448 H for biphenyl 2-hydroxylation; and P-451 II and P-448 H for estradiol 2-hydroxylation. P-451 I, P-450 II, and P-450 III were somewhat poorer catalysts in metabolizing all the substrates except for benzphetamine and aminopyrine, but their substrate specificities were still distinguishable from one another. Of all the purified cytochrome P-450's, P-452 showed the least ability to metabolize all the substrates. Judging from the properties, it appears that six forms in male Wistar rats correspond to the distinct forms of cytochrome P-450 in Long-Evans and/or Sprague-Dawley rats reported by other workers, but P-451 I is a new constitutive isozyme in Wistar rats.     

Sex difference of cytochrome P-450 in the rat: Purification,characterization, and quantitation of constitutive forms of cytochrome P-450 from liver microsomes of male and female rats

One of each constitutive form of cytochrome P-450 from liver microsomes of adult male and female rats was purified essentially following the same method to an apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights estimated by the electrophoresis were 52,000 and 50,000 for forms of cytochrome P-450, P-450-male, and P-450-female, purified from male and female rats, respectively. In addition, the purified preparations of P-450-male and P-450-female showed properties different from each other with respect to spectral characteristics and catalytic activities. In Ouchterlony double diffusion plates, partially purified rabbit immunoglobulin G (IgG) raised against P-450-male and P-450-female showed very weak or no cross-reactivity with P-450-female and P-450-male, respectively. From these results, P-450-male was confirmed to be a form distinct from P-450-female. The anti-P-450-male and anti-P-450-female antibodies, which had been further purified by immunoadsorption, did not form any apparent precipitation bands with liver microsomes from untreated female and male rats, respectively. Supporting this, radial immunodiffusion analysis for P-450-male and P-450-female with an agarose gel impregnated with the rabbit antibodies showed that P-450-male and P-450-female appear in liver microsomes rather specifically depending on the sex hormones. Based on these results, sex differences in drug metabolism in the rat were confirmed as explicable, at least in part, by the presence of distinct forms of cytochrome P-450 in microsomes of male and female rats.     

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus.

A soybean flour-induced, soluble cytochrome P-450 (P-450soy) was purified 130-fold to homogeneity from Streptomyces griseus. Native cytochrome P-450soy is a single polypeptide, with a molecular weight of 47,500, in association with one ferriprotoporphyrin IX prosthetic group. Oxidized P-450soy exhibited visible absorption maxima at 394, 514, and 646 nm, characteristic of a high-spin cytochrome P-450. The CO-reduced difference spectrum of P-450soy had a Soret maximum at 448 nm. When reconstituted with spinach ferredoxin and spinach ferredoxin:NADP+ oxidoreductase, purified cytochrome P-450soy catalyzed the NADPH-dependent oxidation of the xenobiotic substrates precocene II and 7-ethoxycoumarin. In vitro proteolysis of cytochrome P-450soy generated a stable and catalytically active cytochrome P-450, designated P-450soy delta.     

Purification of characterization of a minor form of hepatic microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls

A minor form of hepatic microsomal cytochrome P-450 has been purified to apparent homogeneity from rats treated with the polychlorinated biphenyl mixture, Aroclor 1254. This newly isolated hemoprotein, cytochrome P-450_e, is inducible in rat liver by Aroclor 1254 and phenobarbital, but not by 3-methylcholanthrene. Two other hemoproteins, cytochromes P-450_b and P-450_c, have also been highly purified during the isolation of cytochrome P-450_e based on chromatographic differences among these proteins. By Ouchterlony double-diffusion analysis with antibody to cytochrome P-450_b, highly purified cytochrome P-450_e is immunochemically identical to cytochrome P-450_b but does not cross-react with antibodies prepared against other rat liver cytochromes P-450 (P-450_a, P-450_c, P-450_d) or epoxide hydrolase. Purified cytochrome P-450_e is a single protein-staining band in sodium dodecyl sulfate-polyacrylamide gels with a minimum molecular weight (52,500) slightly greater than cytochromes P-450_b or P-450_d (52,000) but clearly distinct from cytochromes P-450_a (48,000) and P-450_c (56,000). The carbon monoxide-reduced difference spectral peak of cytochrome P-450_e is at 450.6 nm, whereas the peak of cytochrome P-450_b is at 450 nm. Ethyl isocyanide binds to ferrous cytochromes P-450_e and P-450_b to yield two spectral maxima at 455 and 430 nm. At pH 7.4, the 455:430 ratio is 0.7 and 1.4 for cytochromes P-450_b and P-450_e, respectively. Metyrapone binds to reduced cytochromes P-450_e and P-450_b (absorption maximum at 445–446 nm) but not cytochromes P-450_a, P-450_c, or P-450_d. Metabolism of several substrates catalyzed by cytochrome P-450_e or P-450_b reconstituted with NADPH-cytochrome c reductase and dilauroylphosphatidylcholine was compared. The substrate specificity of cytochrome P-450_e usually paralleled that of cytochrome P-450_b except that the rate of metabolism of benzphetamine, benzo[a]pyrene, 7-ethoxycoumarin, hexobarbital, and testosterone at the 16α-position catalyzed by cytochrome P-450_e was only 15–25% that of cytochrome P-450_b. In contrast, cytochrome P-450_e catalyzed the 2-hydroxylation of estradiol-17β more efficiently (threefold) than cytochrome P-450_b. Cytochrome P-450_d, however, catalyzed the metabolism of estradiol-17β at the greatest rate compared to cytochromes P-450_a, P-450_b, P-450_c, or P-450_e. The peptide fragments of cytochromes P-450_e and P-450_b, generated by either proteolytic or chemical digestion of the hemoproteins, were very similar but not identical, indicating that these two proteins show minor structural differences.     

Purification and properties of cytochrome P-450 from untreated monkey liver microsomes

Untreated monkey liver cytochrome P-450 (monkey P-450) has been purified to a specific content of 14.9 n mole/mg protein. The purified preparation was apparently homogeneous and the minimum molecular weight was estimated to be 50,000 by SDS-PAGE. Absolute spectrum of the oxidized form showed peaks at 565, 535 and 417 nm. The monkey P-450 was active in the mixed function oxidation of benzphetamine, aminopyrine, ethylmorphine, aniline and 7-ethoxycoumarin in the presence of rat liver NADPH-cytochrome P-450 reductase and DLPC. Anti monkey P-450 IgG could not inhibit rat P-450s (PB P-450, MC P-448(1) and MC P-448(2] catalyzed 7-ethoxycoumarin O-deethylation activities.     

Purification and characterization of human placental aromatase cytochrome P-450

Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic detergent, Nonidet P-40. The preparation exhibits a single major band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a specific content of 11.5 nmol of P-450/mg of protein. The purified enzyme displays spectroscopic properties typical of the ferric and ferrous forms of cytochrome P-450. Full enzymatic activity can be reconstituted with rabbit liver microsomal cytochrome P-450 reductase and Nonidet P-40. Purified aromatase cytochrome P-450 displays catalytic characteristics similar to the enzyme in intact microsomes in the aromatization of androstenedione, 19-hydroxyandrostenedione and 19-oxoandrostenedione. Testosterone and 16 alpha-hydroxytestosterone are aromatized at maximal rates similar to androstenedione, and all substrates exhibit relative affinities corresponding to those observed in microsomes. We have raised rabbit antibodies to the purified enzyme which show considerable specificity and sensitivity on immunoblots.     

Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions

The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone.     

Heterogeneity of liver microsomal cytochrome P-450: the spectral characterization of reactants with reduced cytochrome P-450.

The aerobic metabolism of benzphetamine by liver microsomes, during a cytochrome P-450-catalyzed mixed-function oxidation reaction, results in the formation of an easily detected spectral complex with an absorption band maximum at 456 nm. Electron paramagnetic resonance studies, as well as studies with the chemical reductant, sodium dithionite, or the oxidant, potassium ferricyanide, indicate that the spectral complex results from the formation of a product adduct with reduced cytochrome P-450. The spectral properties of this product complex of cytochrome P-450 have been compared to those observed with carbon monoxide, metyrapone, and ethylisocyanide. The reaction of these reagents to specific pools of microsomal cytochrome P-450 permits the identification of at least two major and two minor types of cytochrome P-450 in liver microsomes prepared from phenobarbital-treated rats.